This invention relates to DNA associated diseases, such as cancer. In one aspect of this invention, this invention relates to a method for determining the level of cellular DNA repair enzyme activity. In another aspect, this invention relates to a method for monitoring the level of activity of cellular DNA repair enzymes in response to a stress. In another aspect, this invention relates to a method for screening individuals for the predisposition to cancer or other diseases associated with DNA damage. In yet another aspect, this invention relates to a method for screening therapeutic agents which may be useful for treating individuals with a predisposition to or a disease associated with DNA damage.
Most living cells possess systems for recognizing and eliminating DNA damage. As used herein, the term "DNA damage" refers to strand breaks, dimerization, unpaired bases, modified bases, conversion of one base into another resulting in unpaired bases, chromatin unwinding or other modifications, etc. For example, E. coli possesses a variety of enzymes for responding to DNA damage, such as enzymes of the SOS repair system and the various Rec proteins. These enzymes, and others, respond to DNA damage caused by U.V. radiation, chemical mutagens and the like. However, little is known about the mechanism by which the repair systems are activated by DNA damage.
In addition to the prokaryotic enzymes discussed above, eucaryotic and mammalian cells are also known to possess DNA repair enzymes. These enzymes are important in controlling diseases associated with DNA damage, as is clearly shown in the disease Xeroderma pigmentosum (Xp). This recessive disease results in hypersensitivity to sunlight, particularly to ultraviolet radiation. The disease is the result of a faulty excision repair system. Fibroblasts from Xp patients are deficient in the ability to excise and correct thymine dimers and other adducts. The deficit has been shown to be in enzymes that function at the excision step of repair. Another disease correlated with faulty DNA repair is Bloom's disease in which an increased frequency of chromosomal aberrations is seen.
DNA repair synthesis has been studied in cancer patients, see particularly the article by R. W. Pero et al entitled "Reduced Capacity for DNA Repair Synthesis in Patients with or Genetically Predisposed to Colorectal Cancer", JNCI, Vol. 70, No. 5, pp. 867-875, May, 1983, and the article by R. W. Pero et al entitled "Unscheduled DNA Synthesis in Mononuclear Leukocytes from Patients with Colorectal Polyps", Cancer Research, Vol. 45, pp. 3388-3391, July, 1985. These articles are of interest to the practices of this invention as applied to the measurement of the activity of DNA repair enzymes as being an indicator of the predisposition or susceptibility of an individual to colorectal cancer.
DNA damage, as indicated herein, may be caused by a number of agents. For example, oxygen supplied at concentrations greater than those of normal air has long been known to damage plants, animals and aerobic bacteria, see J. D. Ballantine, Pathology of Oxygen Toxicity, 1982, Academic Press, New York. It has been proposed that many of the damaging effects of O.sub.2 could be attributed to the formation of O.sub.2 radicals, see D. L. Gilbert (ed) Oxygen and Living Processes: An Interdisciplinary Approach, 1981, Springer Verlag, New York. The reactive oxygen species are superoxide, H.sub.2 O.sub.2 and a hydroxyl radical. These are generated in vivo, i.e. endogenously in the body, as a consequence of normal metabolism, see B. N. Ames, Science, 221: 1256-1264, 1983. The oxidation of certain cellular components by these oxygen species could, in turn, contribute both to aging and to age-dependent diseases, such as cancer, see P. A. Cerutti, Science, 227:375-380, 1985.
H.sub.2 O.sub.2 is produced by all viable cells, see Romasarma, Biochem.Biophysica Acta, 694:69-93, 1982, and it can be both a mutagen/carcinogen or promoter, see Troll & Wiesner, Ann. Rev. Pharmocol. Toxicol. 25:509-528, 1985, depending upon the cell type. The molecular response of a cell to stress whether it be induced by hyperthermia or by H.sub.2 O.sub.2 is very similar, see Christman et al, Cell 41:753-762, 1985.
The practice of this invention in one embodiment employs H.sub.2 O.sub.2 as an agent for oxidative stress to produce cellular DNA damage, thereby to induce a cellular DNA repair enzyme response, such as a response of the DNA repair enzyme adenosine diphosphate ribosyl transferase (ADPRT).
ADPRT is a nuclear enzyme which covalently attaches ADP-ribose moieties derived from NAD to chromatin proteins, see Hayoishi and Ueda, Ann. Rev. Biochem. 46:96-116, 1977 and Purnell et al Biochem. Soc. Transa. 8:215-227, 1980. The enzyme is dependent on DNA and is strongly stimulated by DNA-strand breaks, see Halldorsson et al FEBS LETT. 85:349-352, 1978; Benjamin and Pill. J. Biol. Chem. 255:10493-10508, 1980; Cohen and Berger, Biochem. Biophys. Res. Commun. 98:268-274, 1981. Although the role of ADPRT in cells is not fully understood, convincing data have been reported in its involvement in DNA repair, see Durkacz et al, Nature 283:593-596, 1980; Zwelling et al Biochem. Biophys. Res. Commun. 104:897-902, 1982; Althaus et al, Biol. Chem. 257:5528-5535, 1982; Chreissen and Shall, Nature 296:271-272, 1982 and Pero et al, Chem. Biol. Interact. 47:265-275, 1983. The involvement of this enzyme in cellular differentiation is reported by Farzaneh et al, Nature 300:262-266, 1982; Johnstone and Williams, Nature 300:368-370, 1982); and Pero et al Carcinogensis 6:1055-1058, 1985. The involvement of this enzyme in gene expression is mentioned by Althaus et al, Nature 300:366-368, 1982 and in connection with longevity by Pero et al, Mutation Res. 142:69-73, 1985. All these cellular events are important to the process of carcinogenesis and thus are important potential regulators of individual sensitivity or risk to develop cancer.
Although, as indicated herein, H.sub.2 O.sub.2 has been known to be produced by viable cells and to have both carcinogenic and promoting properties, it has never been shown to directly activate ADPRT in eucaryotic cells. Moreover, interindividual variation in stress-induced ADPRT, such as oxidative, e.g. H.sub.2 O.sub.2 stress-induced ADPRT, was not known nor was any link to cancer or DNA associated disease susceptibility previously known. In the development of this invention there has been observed in 100 uM H.sub.2 O.sub.2 -induced ADPRT measured values, a greater than 50-fold variation in the cell population tested.
The disclosures of the above-identified publications are herein incorporated and made part of this disclosure.
It is an object of this invention to provide a method whereby individuals with a predisposition to diseases associated with DNA damage could be recognized. Upon recognition, such individuals might then beneficially receive more frequent diagnostic examinations, pretreatment with drugs and the like.
It is also an object of this invention to provide a method for measuring the activity of DNA repair enzymes, particularly ADPRT activity.
It is also an object of this invention to provide a method for screening agents for potential therapeutic value for the treatment of individuals predisposed to diseases associated with the activity of DNA repair enzymes, such as the activity of ADPRT.
How these and other objects of this invention are achieved will become apparent in the light of the accompanying disclosure made with reference to the accompanying drawing which graphically illustrates the relationship of cancer patients and those with a positive family history of cancer or a negative family history of cancer with the measured ADPRT activity of such individuals.